The enzyme 3_oxo-delta5-steroid isomerase (KSI) from Pseudomonas testosteroini is an example of a class of enzymes that catalyze reactions proceeding through an enol(ate) intermediate. An understanding of the mechanism of these enzymes has long been the goal of mechanistic enzymologists. Recently, Gerlt and Gassman have postulated that low barrier hydrogen bonds are important in these and other reactions. While their theory may provide the basis for the understanding of a variety of enzymatic reactions, it has not been tested experimentally. Our recent determination of the complete energetic profile of KSI makes it an excellent enzyme with which to test this theory. We will examine the contribution of hydrogen bonding to the stability of both the enzyme- bound intermediate and the transition state(s) for proton transfer. Experiments will include the determination of the complete free energy profiles for a variety of mutants that involve the replacement of the enzyme electrophile Tyr-14 with a series of fluorotyrosines. This work will enable us to determine the Bronsted coefficients for both the internal equilibrium and the kinetic constants. These results will be interpreted in terms of the type of hydrogen bonding that is involved in these processes. In addition, we will examine the free energy profiles for a series of mutants of the phenylalanines at positions 101, 30, 80 and 103. Mutants with amino acids at position 101 (Fl10L, F101A) that are less hydrophobic that Phe have been shown to be much less active than wild type KSI, suggesting that there is also a hydrophobic component to the catalysis by KSI. The free energy profiles for these mutants should allow us to determine the mechanism by which this catalysis occurs.